Abstract
MicroRNAs (miRNAs) are small RNA molecules of 21–25 nucleotides that regulate cell behavior through inhibition of translation from mRNA to protein, promotion of mRNA degradation and control of gene transcription. In this study, we investigated the miRNA expression signatures of cell cultures undergoing osteoblastic and osteocytic differentiation from mesenchymal stem cells (MSC) using mouse MSC line KUSA-A1 and human MSCs. Ninety types of miRNA were quantified during osteoblastic/osteocytic differentiation in KUSA-A1 cells utilizing miRNA PCR arrays. Coincidently with mRNA induction of the osteoblastic and osteocytic markers, the expression levels of several dozen miRNAs including miR-30 family, let-7 family, miR-21, miR-16, miR-155, miR-322 and Snord85 were changed during the differentiation process. These miRNAs were predicted to recognize osteogenic differentiation-, stemness-, epinegetics-, and cell cycle-related mRNAs, and were thus designated OstemiR. Among those OstemiR, the miR-30 family was classified into miR-30b/c and miR-30a/d/e groups on the basis of expression patterns during osteogenesis as well as mature miRNA structures. In silico prediction and subsequent qRT-PCR in stable miR-30d transfectants clarified that context-dependent targeting of miR-30d on known regulators of bone formation including osteopontin/spp1, lifr, ccn2/ctgf, ccn1/cyr61, runx2, sox9 as well as novel key factors including lin28a, hnrnpa3, hspa5/grp78, eed and pcgf5. In addition, knockdown of human OstemiR miR-541 increased Osteopontin/SPP1 expression and calcification in hMSC osteoblastic differentiation, indicating that miR-541 is a negative regulator of osteoblastic differentiation. These observations indicate stage-specific roles of OstemiR especially miR-541 and the miR-30 family on novel targets in osteogenesis.
Highlights
RNA transcripts from the genome spontaneously form stem, loop and/or bulge structures, and many of these have been reported to act as primary miRNA
After adding osteogenic differentiation supplements to cells, calcium was gradually deposited in KUSA-A1 and hMSC culture until 2 weeks after the addition (Fig. 1)
Mesenchymal cell markers ctgf/ccn2, cyr61/ccn1 and nov/ccn3 were induced at initial stimulation (4 hrs) but declined as cells differentiated along the osteocyte lineage (4w+: Fig. 2A)
Summary
RNA transcripts from the genome spontaneously form stem, loop and/or bulge structures, and many of these have been reported to act as primary miRNA (pri-miRNA). An RNase III in mammalian, cuts the pri-miRNA to create premiRNA that has a simpler stem-bulge structure. Exportin-5 subsequently exports the pre-miRNA to the cytoplasm [1,2]. Dicer, another RNase III, cuts the pre-miRNA and creates double strand RNA (dsRNA). Each strand of the dsRNA is a 21– 25mer nucleotide, and acts as miRNA by forming the RNAinduced silencing complex (RISC) along with the Argonaute protein [3]. MiRNA have been detected in exosomes suggesting a potential role in regulating gene expression in a paracrine or autocrine manner [10]. Recent studies imply that miRNA may be involved in a feedback system of gene expression where the miR-371-373 cluster was controlled by Wnt target beta-catenin/TCF and this miRNA cluster could inhibit DKK1, a Wnt/LRP inhibitor [11]
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